Transforming growth factor beta (TGFβ) isoforms (β1, β2, and β3) are homodimeric polypeptides of 25 kDa. They are secreted in a latent form and only a small percentage of total secreted TGFβs are activated under physiological conditions. TGFβ binds to three different cell surface receptors called type I (RI), type II (RII), and type III (RIII) receptors. RI and RII are serine/threonine kinase receptors. RIII (also called betaglycan) has two TGFβ binding sites in its extracellular domain, which are called the E and U domains (BGE and BGU, respectively). TGFβ1 and TGFβ3 bind RII with an affinity that is 200-300 fold higher than TGF-β2 (Baardsnes et al., Biochemistry, 48, 2146-55, 2009); accordingly, cells deficient in RIII are 200- to 300-fold less responsive to equivalent concentrations of TGF-β2 compared to TGF-β1 and TGFβ-3 (Chiefetz, et al (1990) J. Bio. Chem., 265, 20533-20538). However, in the presence of RIII, cells respond roughly equally to all three TGF-β isoforms, consistent with reports that show that RIII can sequester and present the ligand to RII to augment TGFβ activity when it is membrane-bound (Chen et al., J. Biol. Chem. 272, 12862-12867, 1997; Lopez-Casillas et al., Cell 73, 1435-1444, 1993; Wang et al., Cell 67, 797-805, 1991; Fukushima et al., J. Biol. Chem. 268, 22710-22715, 1993; Lopez-Casillas et al., J. Cell Biol. 124, 557-568, 1994). Binding of TGFβ to RII recruits and activates RI through phosphorylation (Wrana et al., Nature 370, 341-347, 1994). The activated RI phosphorylates intracellular Smad2 and Smad3, which then interact with Smad4 to regulate gene expression in the nucleus (Piek et al., FASEB J. 13, 2105-2124, 1999; Massague and Chen, Genes & Development 14, 627-644, 2000). Through its regulation of gene expression, TGFβ has been shown to influence many cellular functions such as cell proliferation, cell differentiation, cell-cell and cell-matrix adhesion, cell motility, and activation of lymphocytes (Massague, Ann. Rev. Cell Biol. 6, 597-641, 1990; Roberts and Sporn, The transforming growth factor-betas. In Peptide growth factors and their receptors I, Sporn and Roberts, eds. (Heidelberg: Springer-Verlag), pp. 419-472, 1991). TGFβ has also been shown or implicated in inducing or mediating the progression of many diseases such as osteoporosis, hypertension, atherosclerosis, hepatic cirrhosis and fibrotic diseases of the kidney, liver, and lung (Blobe et al., N. Engl. J. Med. 342, 1350-1358, 2000). Perhaps, the most extensively studied function of TGFβ is its role in tumor progression.
TGFβs have been shown to be potent growth inhibitors in various cell types including epithelial cells (Lyons and Moses, Eur. J. Biochem. 187, 467-473, 1990). The mechanism of the growth inhibition by TGFβ is mainly due to the regulation of cell cycle-related proteins (Derynck, Trends. Biochem. Sci. 19, 548-553, 1994; Miyazono et al., Semin. Cell Biol. 5, 389-398, 1994). Thus, aberrant regulation of cell cycle machinery such as loss of retinoblastoma gene product during tumorigenesis can lead to loss of growth inhibition by TGFβ. Furthermore, mutational inactivation of TGFβ receptors, Smad2, and Smad4 has been reported in various carcinomas (Massague et al., Cell 103, 295-309, 2000). For example, loss of RI and/or RII expression is often observed in some human gastrointestinal cancers (Markowitz and Roberts, Cytokine, Growth Factor, Rev. 7, 93-102, 1996).
While many carcinoma cells lose response to TGFβ's growth inhibition, they often overproduce active TGFβ isoforms when compared to their normal counterpart (Reiss, Microbes and Infection 1, 1327-1347, 1999). This is likely to result in the selection of cancer cells that are resistant to TGFβ's growth inhibitory activity. Indeed, an increased level of TGFβ1 is strongly associated with the progression of many types of malignancies and poor clinical outcome (Reiss, Microbes and Infection 1, 1327-1347, 1999). For example, serum TGFβ1 levels have been shown to correlate to tumor burden, metastasis, and serum prostate specific antigen (PSA) in prostate cancer patients (Adler et al., J. Urol. 161, 182-187, 1999; Shariat et al., J. Clin. Oncol. 19, 2856-2864, 2001). Consistent with these observations, marked increase of TGFβ1 and TGFβ2 expression was observed in an aggressive androgen-independent human prostate cancer cell line when compared to its less aggressive androgen-dependent parent cell line, LNCap (Patel et al., J. Urol. 164, 1420-1425, 2000).
Several mechanisms are believed to mediate TGFβ's tumor-promoting activity (Arteaga et al., Breast Cancer Res. Treat. 38, 49-56, 1996; Reiss, Microbes and Infection 1, 1327-1347, 1999). TGFβ is a potent immune suppressor (Sosroseno and Herminajeng, Br. J. Biomed. Sci. 52, 142-148, 1995). Overexpression of TGFβ1 in the rat prostate cancer cells was associated with a reduced immune response during tumor formation suggesting that TGFβ may suppress host immune response to the growing tumor (Lee et al., Prostate 39, 285-290, 1999). TGFβ has also been shown to be angiogenic in vivo (Fajardo et al., Lab. Invest. 74, 600-608, 1996; Yang and Moses, J. Cell Biol. 111, 731-741, 1990; Wang et al., Proc. Natl. Acad. Sci. U.S.A. 96, 8483-8488, 1999). Overexpression of TGFβ during cancer progression is often associated with increased angiogenesis and metastasis suggesting that TGFβ may promote metastasis by stimulating tumor blood vessel formation (Roberts and Wakefield, Proc. Natl. Acad. Sci. U.S.A. 100, 8621-8623, 2003). TGFβ also plays an important role in promoting bone metastasis of human prostate and breast cancers (Koeneman et al., Prostate 39, 246-261, 1999; Yin et al., J. Clin. Invest 103, 197-206, 1999). Both TGFβ1 and TGFβ2 are produced by bone tissue, which is the largest source of TGFβ in the body (Bonewald and Mundy, Clin. Orthop. 261-276, 1990). The latent TGFβ can be activated by proteases such as PSA and urokinase plasminogen activator, which are abundantly secreted by cancer cells (Koeneman et al., Prostate 39, 246-261, 1999). Taken together, TGFβ can act in tumor microenvironment to promote carcinoma growth, angiogenesis, and metastasis.
Because of its involvement in the progression of various diseases, TGFβ has been targeted for the development of novel therapeutic strategies. One way of antagonizing TGFβ activity is to utilize the ectodomain of TGFβ type II receptor or type III receptor (betaglycan (BG)). It has previously been shown that ectopic expression of a soluble RIII (sBG) in human carcinoma cell lines can significantly inhibit tumor growth, angiogenesis, and metastasis when they are inoculated in athymic nude mice (Bandyopadhyay et al., Cancer Res. 59, 5041-5046, 1999; Bandyopadhyay et al., Oncogene 21, 3541-3551, 2002b). More recently, it has been shown that systemic administration of recombinant sRIII can inhibit the growth, angiogenesis, and metastasis of the xenografts of human breast carcinoma MDA-MB-231 cells in nude mice (Bandyopadhyay et al., Cancer Res. 62, 4690-4695, 2002a). However, the inhibition was only partial. This could be due, in part, to the fact that the cells produced active TGFβ1 and active TGFβ2 and the anti-TGFβ potency of sRIII is 10-fold lower for TGFβ1 than for TGFβ2 (Vilchis-Landeros et al., Biochem. J. 355, 215-222, 2001). Interestingly, while the extracellular domain of RII (sRII) has very low affinity for TGFβ2, its affinity for TGFβ1 and TGFβ3 is more than ten times higher than that of sRIII (Lin et al., J. Biol. Chem. 270, 2747-2754, 1995; Vilchis-Landeros et al., Biochem. J. 355, 215-222, 2001).
While numerous TGFβ antagonists have been prepared and tested, all have less than complete TGFβ isoform inhibiting properties. Thus, there is a need for additional TGF antagonists or inhibitors.